Fluorometers

ABSTRACT

In apparatus for the production and detection of fluorescence at a sample surface, the height of the apparatus above the sample surface is reduced, and loss of the emitted fluorescence due to reflection loss and light scattering is minimized. The apparatus comprises a three-dimensionally curved light reflecting surface ( 40 ) that directs light from a light source ( 32 ) transversely to its original path and focuses the light on to an illumination zone ( 30 ) at or below the sample surface. The reflecting surface ( 40 ) also collects, directs and at least partially collimates emitted fluorescence transversely to its original path and towards a detector ( 46 ).

The present invention relates to fluorometers, being apparatus forproducing and measuring fluorescence, whether using intensity or timeresolved measurements.

Epifluorescence microscopes conventionally have a linear opticalarrangement in which a sample location, beam splitter and detector arearranged spaced, for instance vertically, along a common axis in a firstdirection, with an excitation light source off to one side. Thisarrangement dictates a minimum height constraint so as to provide roomfor fluorescence light emitted from a sample location to be collimatedby a lens system, passed through the beam splitter, filtered to removewavelengths other than that of the fluorescence and finally to befocussed onto the detector. Such a conventional arrangement isillustrated in FIG. 1 as discussed in greater detail below. Influorescence applications the intensity of the emitted fluorescence isusually weak. Furthermore, the fluorescence is emitted isotropically oraccording to a Lambertian radiation pattern if the fluorophore issituated in a light scattering medium such as skin. In both cases thedetected fluorescence increases with the numerical aperture of theoptical system. In the common epifluorescence set-up a large numericalaperture is normally obtained by the use of a number of lenses, whichintroduces reflection loss and light scattering even for coated lenses.

It would be desirable to develop an alternative general opticalarrangement with reduced complexity that can be used to reduce thenecessary height of the apparatus measured from a sample location.

Accordingly, the present invention now provides in a first aspectapparatus for the production and detection of fluorescence at or below asurface, said apparatus comprising:

-   -   a light source for directing fluorescence excitation light along        a light path extending over a said surface;    -   a reflector having a three dimensionally curved, shell-like        light reflecting interface positioned to receive light from the        light source passing over said surface along a portion of said        light path and to reflect said light transversely with respect        to said portion of the light path so as to focus said light on        an illumination zone at or below said surface for stimulation of        fluorescence at said zone, and to collect fluorescence light        emitted at said zone and to reflect and at least partially        collimate said light to pass back along said portion of the        light path; and    -   a detector for receiving said light emitted as fluorescence        after reflection at said interface.

The ‘surface’ referred to above may be a physical surface defined by aninterface between two different materials or may be a virtual surfacedefinable with respect to the apparatus.

The apparatus may further comprise a beam splitter reflecting lightemitted by said light source to pass to said reflector and receivingfluorescence light from said reflector and passing said fluorescencelight to said detector.

Said reflector, light source and detector are preferably arranged in agenerally coplanar manner and said beam splitter preferably has a planarreflective interface that lies in a plane orthogonal to the co-planarityof the reflector, light source and detector.

The apparatus may further comprise an excitation filter selecting anexcitation wavelength from the light emitted by the light source to passto said reflector.

The apparatus may further comprise an emission filter selecting anemitted fluorescence wavelength to pass to said detector.

The apparatus may comprise a lens focussing fluorescence light on saiddetector. Alternatively, a further focussing reflective interface may beused.

The or each said reflector interface may substantially have the form ofa partial paraboloid, aspheric, toroidal, or biconic surface. Such areflector interface may be paraboloid, aspheric, toroidal, or biconicsurface. Spheric surfaces and aspheric surfaces such as hyperbolas,parabolas, ellipsoids and oblate ellipsoid reflector interfaces may bedefined by an equation

$Z = \frac{{cr}^{2}}{1 + \sqrt{1 - {\left( {1 + k} \right)c^{2}r^{2}}}}$

wherein:c is from 0.07 to 0.5 and k is from −1.5 to −0.7, where z is the “sag”of z-coordinate along the rotational axis, c is the curvature (thereciprocal of the radius R), k is the conical constant and r is theradial coordinate. Other surfaces may be described by similar equationsinvolving an added Taylor expansion. Similarly, equations are availablefor toroids, e.g. faceted toroids, or piecewise linear toroids and forbiconic surfaces.

The reflector interface of the reflector directing light to theillumination zone may include that part of a paraboloid, aspheric,toroidal, or biconic surface that is generated by the cutting of aparaboloid, aspheric, toroidal, or biconic surface by a right circularcylinder erected centred on the illumination zone.

Said reflector interface may preferably substantially have the form of apart of a half paraboloid.

The apparatus may further include a housing containing the light source,reflector and detector and having a base surface containing a window forpassing excitation light out of the housing and receiving fluorescencelight into the housing and being for engagement in use against the saidsurface at or below which said fluorescence occurs.

Preferably, said light path makes an angle of no more than 10 degreeswith a plane defined by said base surface.

The invention will be further described and illustrated with referenceto the accompanying drawings in which:

FIG. 1 is a schematic side elevation view of a conventionalepifluorescence measuring apparatus;

FIG. 2 is a perspective view of a first embodiment of apparatusaccording to the present invention;

FIG. 3 is an illustration showing the zone of a parabolic mirror inwhich most energy is collected when light is emitted from a surface overwhich the mirror lies;

FIG. 4 is a plan view from above of a second embodiment according to theinvention;

FIG. 5 is a perspective view of the apparatus of FIG. 4;

FIG. 6 is a cross section on the line VI-VI′ marked in FIG. 4;

FIG. 7 shows a modification of the apparatus of FIG. 4;

FIG. 8 shows a third embodiment according to the invention in plan fromabove;

FIG. 9 is a graph of the variation in shape of a parabolic mirror withthe conical constant; and

FIG. 10 is a graph showing the variation in the ratio of detectedoptical power and illumination power with changing conical constant.

A common set up for epifluorescence measurement is shown in FIG. 1. Itis designed for producing and detecting fluorescence at a samplelocation 10, which might be for instance at or below the surface of abody or article, e.g. on a microscope slide. Typically such a surfacewill be horizontal and for convenience, this is assumed in the followingdescription of the apparatus. An excitation light source 12, such as anLED, is positioned off to one side of the sample location 10 and at aheight h above the sample location. The LED emits excitation light 16which is passed through an excitation filter 14 to select a desiredexcitation wavelength. The excitation light falls on an angled beamsplitter 18 such as a parallel sided dichroic beam splitter 18. Aportion of the excitation light is reflected down at right anglestowards the sample location and passes through a focussing lens system20 comprising one or more simple or compound lenses.

Fluorescence and reflected excitation light emanating from the samplelocation are captured by the lens system 20 and collimated into aparallel beam which passes through the beam splitter 18 to an emissionfilter 22, which ideally removes all but the fluorescence wavelength,and from there to a second lens system 24 that focuses the fluorescenceonto a detector 26. Like the lens system 20, the lens system 24 maycomprise one or more simple or compound lenses.

The whole device has a total height above the sample of h′, which isaround 3×h.

It has been proposed to monitor concentrations of chemical species inthe human or animal body by fluorescence based techniques. These wouldinvolve directing the excitation light onto the skin and detectingfluorescence emission from the skin. Relevant teachings includeWO00/02048 and WO02/30275.

We have noted that it would be desirable in such circumstances andothers to reduce the dimension h′ of the fluorescence detectionapparatus used.

FIG. 2 shows an embodiment of apparatus according to the invention.Here, the apparatus is depicted placed on the surface of the skin or asimilar surface on which fluorescence is to be detected. For conveniencein the following description, the plane of the skin will be treated asbeing horizontal. A light source 32 emits light along a path parallel tothe skin surface, and determines a height h for the apparatus whichapproximately equals to the total height h′ of the device.

The light passes through an excitation filter 34 to a dichroic beamsplitter 38 disposed in a vertical plane and is diverted towards a halfparaboloid shaped mirror reflector 40 which replaces the lens system 20of the conventional apparatus. The reflector focuses the light down ontoan illumination zone 30 on the skin surface or towards an illuminationzone just below the skin surface. The reflector collects and collimatesfluorescence emissions from the illumination zone and directs theemitted light back to the beam splitter, which it passes through toreach an emission filter 42. From there, the light is focused by a lenssystem 44 of the same type as lens system 24 and is detected at adetector 46.

It can be seen that the vertical height of the apparatus has beenreduced from approximately 3×h in FIG. 1 to just h in FIG. 2.

Generally, interference filters are used in fluorometers as theexcitation and emission filters. These require that the incident lightbe orthogonal to the plane of the filter if the expected wavelengthpassing properties are to be obtained, because with obliquely incidentlight, the filter pass band will be shifted towards shorter wavelengths.Collimation of the light beams incident on these filters is thereforerequired.

The paraboloid reflector 40 may be constructed in a number of ways toproduce a reflecting interface of the desired shape. For instance, theinterface may be between air inside the paraboloid and a reflectiveconcave interior surface of a body. Alternatively, the interface may beformed at the convex exterior surface of a solid hemi-paraboloid member,for instance a silvered exterior of a glass or plastics hemi-paraboloidblock.

The whole of the illustrated apparatus will be bounded by a housing orcasing (as seen in FIG. 6) having a bottom face containing an aperturewhich may be filled with a transparent window (suitably of glass, silicaor in some applications plastics) for protecting the optics against dustand humidity and for allowing the passage of excitation and fluorescencelight to and from a sample in the illumination zone. Said apparatus maythus be a simple opening in the casing or an optical window.

Signals from the detector may be fed to suitable electronic circuitryfor analysis in a known manner. The LED may be powered by suitableelectronic circuitry as known and the LED supply circuitry and thedetected signal processing circuitry will generally form part of anintegral circuitry for producing intensity based or time resolved(frequency domain) measurements of the fluorescence, e.g. in a FRET(fluorescence resonance energy transfer) based assay.

The illustrated apparatus provides a high numerical aperture for thecapture of fluorescence from the illumination zone whilst enabling acompact optical geometry suited for use where space is at a premium,e.g. in a device to be worn on the body.

Not all of the illustrated paraboloid surface is needed in order toobtain good results. The paraboloid need not, as shown, be arranged withits axis coincident with the surface on which the device is to be usedbut may be angled up to gain some additional height above the surfacefor the detector optics. The beam of fluorescent light may not becollimated entirely by the reflector but may in part be collimated by anauxiliary lens system. The physical illumination zone of the apparatusneed not lie at the focus of the reflector. The reflector interface neednot be in the form of a true paraboloid. These concepts are furtherillustrated in subsequent Figures.

As shown in FIG. 3, the area of the paraboloid surface M that willreceive and collimate out to the detector most of the fluorescenceemitted from the illumination zone will be that defined by theintersection of a right circular cylinder C centred on the illuminationzone with the paraboloid surface itself. The remainder of the paraboloidsurface need not therefore be present. Clearly, the smaller thecylinder, the less light will be captured, so preferably at least asmuch of the paraboloid is present as is defined by the intersection witha cylinder of a radius r not less than 50%, preferably not less than75%, more preferably not less than 90% of the distance marked f from theorigin to the focal point of the paraboloid. The radius r of thecylinder need not be smaller than the focal length of the parabolicmirror, as in the case shown in FIG. 3, but can be larger.

Whilst in FIG. 2 a lens system is shown focusing the light onto thedetector, it will be appreciated that this could also be a reflectorsystem instead, which might suitably resemble that used to focus theexcitation light on the illumination zone. Such a system is shown inFIGS. 4 to 6.

In the illustrated apparatus, the illustrated components are as in FIG.2 except that the lens system 44 is replaced by a part parabolic mirror45 which resembles mirror 40 but is arranged to focus the fluorescencelight to the side where the detector 46 is now positioned. As seen inFIG. 6, the apparatus comprises a housing 50 having a base plate 52lying on the skin and containing a window 54 as previously describedwhich defines the illumination zone 30. The housing comprises upper andlower half shells having internal formations to cradle and support theoptical components, the shells being secured together by screws asshown.

As shown in FIG. 7, the base surface of the housing may lie at an angleα, preferably not exceeding 10°, more preferably not exceeding 5°, tothe axis of the paraboloid surface. This will give some additional spacefor the optical and electronic components but will also result in someadditional height at the detector end of the apparatus. Thismodification may be employed both in relation to the embodiment usingmirrors at each end as shown and in relation to the embodiment of FIG.2. Furthermore, the focal point of the mirror 40 may differ from thelocation of the sample, as shown on FIG. 7 by the distance 5, in orderto compensate for the radiation pattern and scattering properties of thesample or surrounding media as in the case of detecting fluorescenceunder the skin.

As shown on FIG. 8, an auxiliary lens system comprising one or moresimple or compound lenses 41 positioned in front of mirror 40 may beused to produce further collimation.

Similarly, the shape of the reflector may deviate from that of aparaboloid in order to accommodate to the radiation pattern andscattering properties of the sample or surrounding media as in the caseof detecting fluorescence under the skin. If this leads to an incompletecollimation of the fluorescence emissions, further collimation may becarried out using an auxiliary lens system 41 as illustrated.

Alternative curved surfaces that may be used include toroidal, asphericand biconic surfaces.

For an aspheric surface defined by

$Z = \frac{{cr}^{2}}{1 + \sqrt{1 - {\left( {1 + k} \right)c^{2}r^{2}}}}$

c is preferably within the range of 0.07 to 0.5 and k is preferablywithin the range of −1.5 to −0.7, where z is the “sag” of z-coordinatealong the rotational axis, c is the curvature (the reciprocal of theradius R) and k is the conical constant and r is the radial coordinate.The shape of the mirror is shown in FIG. 9 for various values of theconical constant.

In FIG. 10 the influence of the conical constant on the detected opticalpower is shown.

The illumination zone need not be a point or a circular area but by theeffect of the use of non-paraboloid reflector shapes may be elongatedeither transversely or longitudinally of the axis of the reflector.

In this specification, unless expressly otherwise indicated, the word‘or’ is used in the sense of an operator that returns a true value wheneither or both of the stated conditions is met, as opposed to theoperator ‘exclusive or’ which requires that only one of the conditionsis met. The word ‘comprising’ is used in the sense of ‘including’ ratherthan in to mean ‘consisting of’.

1-12. (canceled)
 13. A method for producing and detecting fluorescence at or below a surface, said method comprising: using a light source, directing fluorescence excitation light along a light path extending over said surface; receiving the excitation light along a portion of said light path with a reflector interface configured to a) reflect said light transversely with respect to said portion of the light path so as to focus said excitation light on an illumination zone at or below said surface for stimulation of fluorescence at said zone, b) collect fluorescence light emitted at said zone and c) reflect said light to pass back along said portion of the light path; and using a detector, receiving said light emitted as fluorescence after reflection at said interface; in which the above components are used adjacent to or worn on a human or animal body.
 14. The method as claimed in claim 13, wherein the reflector interface is three dimensionally curved.
 15. The method as claimed in claim 13, wherein the light source, detector and interface are provided in a housing configured to be worn on the body, and the method further comprises placing the housing adjacent said surface.
 16. The method as claimed in claim 13, further comprising extending the whole of the light paths from the light source to the interface and from the interface to the detector extend over the said surface.
 17. The method as claimed in claim 13, further comprising, using a beam splitter, reflecting light emitted by said light source to pass to said interface and receiving fluorescence light from said interface and passing said fluorescence light to said detector.
 18. The method as claimed in claim 17, further comprising arranging said interface, light source and detector in a generally coplanar manner, and providing said beam splitter with a planar reflective interface that lies in a plane orthogonal to the co-planarity of the reflector, light source and detector.
 19. The method as claimed in claim 13, further comprising selecting an excitation wavelength from the light emitted by the light source to pass to said interface.
 20. The method as claimed in claim 13, further comprising selecting an emitted fluorescence wavelength to pass to said detector.
 21. The method as claimed in claim 13, further comprising focusing fluorescence light on said detector.
 22. The method as claimed in claim 13, further comprising forming said interface substantially in the shape of a partial paraboloid, aspheric, toroidal, or biconic surface.
 23. The method as claimed in claim 22, wherein said reflector interface is in a form obtained by cutting said partial paraboloid, aspheric, toroidal, or biconic surface by a right circular cylinder erected centered on the illumination zone.
 24. The method as claimed in claim 13, further comprising shaping said reflector interface substantially in the form of a part of a half paraboloid.
 25. The method as claimed in claim 15, wherein the housing includes a base surface containing a window, and wherein the method further comprises passing excitation light out of the housing and receiving fluorescence light into the housing.
 26. The method as claimed in claim 25, further comprising providing said light path at an angle of no more than 10 degrees with a plane defined by said base surface.
 27. The method as claimed in claim 13, further comprising fixing the relative positions of the light source, detector and interface.
 28. A method for producing and detecting fluorescence at or below a human or animal's skin, said method comprising: directing fluorescence excitation light along a light path extending over the skin in use; reflecting said excitation light transversely with respect to the skin for stimulation of fluorescence; directing any collected fluorescence light along a path extending over the skin; and detecting said collected fluorescence light.
 29. The method as claimed in claim 28, wherein the excitation light and/or the collected fluorescence light extend substantially parallel to or not substantially perpendicular relative to the surface.
 30. Apparatus for producing and detecting fluorescence at or below a surface, said apparatus comprising: a light source to direct fluorescence excitation light along a light path extending over to said surface; a fixed reflector to reflect the excitation light transversely with respect to said surface for stimulation of fluorescence, to collect fluorescence light emitted, and to reflect said fluorescence light to pass back along a path extending over said surface; and a detector to receive said light emitted as fluorescence.
 31. The apparatus as claimed in claim 30, further comprising providing a common housing having said light source, reflector and a detector.
 32. The apparatus as claimed in claim 31, wherein said common housing is configured to be mounted on a human or animal body.
 33. The apparatus as claimed in claim 31, wherein said common housing includes a window wherein said window extends in a plane that extends over the light path of the excitation light.
 34. The apparatus as claimed in claim 30, wherein the reflector, detector and light source are fixed relative to one another.
 35. The apparatus as claimed in claim 30, wherein the excitation light and/or the collected fluorescence light extend substantially parallel to or not substantially perpendicular relative to the surface.
 36. The apparatus as claimed in claim 13, wherein the light source, reflector and detector are provided in a substantially common plane extending substantially parallel to the surface. 